The green fluorescent protein (GFP) from jellyfish Aequorea victoria and homologues fluorescent proteins (FPs) from Anthozoa corals have become invaluable tools for cell imaging. Anthozoa FPs is available in colors and with features unlike those of GFP variants and, thus, provides powerful templates for new probes for molecular labeling and intracellular detection. Several Anthozoa FPs have been already developed into biotechnological tools. Nevertheless, the continuing progress in optical microscopy methods and fluorescence imaging approaches requires probes with new colors and photochemical properties. Two super-resolution fluorescence techniques, stimulated emission depletion (STED) fluorescence microscopy and photoactivated localization microscopy (PALM), have been recently developed. With the improvement of two-photon lasers, a deep-tissue intravital imaging in live animals has become widely available. However, enhanced monomeric FPs suitable for these imaging techniques exist in two colors only. Our analysis of the chromophore formation mechanisms in Anthozoa FPs suggest that fluorescent probes with novel spectral and photochemical features can be indeed designed. On the basis of existing monomeric FPs we plan to develop three new types of protein labels complementary to the available green and red probes. These include monomeric photoactivatable FPs (PA-FPs), which are initially dark but become fluorescent in Blue, Orange or Far-red regions upon irradiation with violet light (Aim 1); monomeric FPs with large Stokes shift (LSS) emission (LSSFPs), which absorb in cyan but fluoresce in Orange or Far-Red regions, and which we further plan to convert into photoactivatable LSS-FPs (Aim 2); and an enhanced monomeric Far-Red FP with improved brightness and further shifted towards far-red for efficient excitation using red lasers (Aim 3). We will apply directed molecular evolution techniques consisting of rational structure-based design and random mutagenesis of candidate proteins, followed by flow cytometry and multiwell plate screening. Moreover, screening methods utilizing two-photon excitation and single-molecule characterization will be developed to optimize photophysical properties of LSS-FPs for intravital imaging and of PA-FPs for PALM, respectively. We will correlate the mutagenesis process with spectral and photochemical changes, in order to gain insight into the molecular evolution of chromophore structures responsible for fluorescence properties and will apply these to the next rounds of molecular evolution. The fluorescent variants will be thoroughly characterized in vitro and as fusion tags in live cells, using a conventional fluorescence microscopy, as well as the super-resolution imaging techniques. The anticipated end result of the proposed research is a collection of molecular fluorescent tools with new fluorescent colors that will be as versatile as the respective green and red proteins. The resulting probes will expand the PA-FP technology to allow diffraction-limited or super-resolution PALM imaging of localization and dynamics of several intracellular proteins simultaneously. The enhanced FRFP and new LSSFPs, excitable with a single wavelength two-photon laser, will further advance the deep-tissue and multicolor intravital imaging approaches.